Restriction-modification (R-M) systems comprise the innate immune system in bacteria and archaea. Their discovery ~50 years ago by Arber, Nathans, and Smith (1978 Nobel Prize in Physiology & Medicine) opened the doors of modern biotechnology. Without R-M enzymes there would haven been no recombinant DNA revolution and no gene technology, as we know it today. R-M systems range from simple Type II enzymes to more complex families of enzymes that require ATP (Type I and III) or that encode both endonuclease and methylation activities within the same polypeptide (Type IIL). Much has been learned over the past two decades about the structure and mechanism of the simple Type II enzymes (such as BamHI and FokI), providing fundamental insights into the basis of extreme protein-DNA selectivity and lending to the creation of novel chimeric nucleases. However, much remains to be learned about the other more complex families of R-M enzymes. EcoP15I is a prototype of the Type III R-M family that functions as a pseudo-helicase or a molecular switch to communicate between distant DNA sites. The DNA is cleaved when two EcoP15I complexes collide. Although Ecop15I was discovered >40 years ago there had been no structural information. We have resolved the crystal structure of the complete Ecop15I complex. We will carry out additional structural and functional studies aimed at understanding its mechanism of translocation and DNA cleavage. MmeI is a prototype of the Type IIL R-M family that provides a natural platform for engineering new DNA-binding specificities. Some success has already been achieved in this direction. We will use structural information on MmeI-like enzymes to identify specificity determinants, which can then be rationally mutated to generate new nucleases. We also look to understand how these enzymes control their nuclease activity, as a means to prevent self-restriction while at the same time allowing for restriction of viral DNA. Overall, we will uncover new structural principles by which these complex R-M systems communicate and cleave DNA over long distances and how specificity determinants can be molded to create new enzymes.